Prevalence of the coagulation factor XIII polymorphism Val34Leu in women with recurrent miscarriage

BACKGROUND: Recurrent miscarriage (RM) syndrome is not an uncommon obstetrical problem of multifactorial etiology. We investigated the role of the coagulation factor XIII (FXIII) Val34Leu polymorphism in RM. METHODS: We recruited 80 subjects (40 normal and 40 with history of RM; of each group 20 pregnant and 20 non-pregnant) and analyzed the prevalence of this polymorphism. The women recruited for the present study had similar age and did not have history of any hemostatic disorders. FXIII levels and activity and the rate of fibrin cross-linking by FXIII genotype Val34Val and Val34Leu were studied. RESULTS: Genotype analyses of patients and normal revealed that the frequencies distribution of Val/Val and Val/leu were statistically similar (P<0.05): 62.5% and 60%, and 37.5% and 40%, respectively; no Leu/leu genotype was found. The FXIII-A subunit levels and activity were also found similar between Val/Val and Val/leu genotypes in the different groups, pregnant and non-pregnant, normal or with RM. The rate of FXIII alpha and gamma-chains fibrin cross-linking was not different between the 2 genotypes. CONCLUSION: From our results we conclude that FXIII Val34Leu polymorphism does not appear to be associated to RM.     

Rapid detection and differentiation of human pathogenic orthopox viruses by a fluorescence resonance energy transfer real-time PCR assay

BACKGROUND: The orthopox viruses that are pathogenic for humans include variola major virus (VAR), monkeypox virus (MPV), cowpox virus (CPV), and to a lesser extent, camelpox virus (CML) and vaccinia virus (VAC). PCR is a powerful tool to detect and differentiate orthopox viruses, and real-time PCR has the further advantages of rapid turnaround time, low risk of contamination, capability of strain differentiation, and use of multiplexed probes. METHODS: We used real-time PCR with fluorescence resonance energy transfer technology to simultaneously detect and differentiate VAR, MPV, CPV/VAC, and CML. An internal control generated by cloning and mutating the PCR target gene facilitated monitoring of PCR inhibition in each individual test reaction. RESULTS: Strain differentiation results showed little interassay variability (CV, 0.4-0.6%), and the test was 100-fold more sensitive than virus culture on Vero cells. Low copy numbers of DNA could be detected with > or =95% probability (235-849 genome copies/mL of plasma). CONCLUSIONS: The real-time PCR assay can detect and differentiate human pathogenic orthopox viruses. The use of an internal control qualifies the assay for high sample throughput, as is likely to be needed in situations of suspected acts of biological terrorism, e.g., use of VAR.     

Mass spectrometric phenotyping of Val34Leu polymorphism of blood coagulation factor XIII by differential peptide display

BACKGROUND: The Val34Leu mutation in the activation peptide of factor XIII (FXIIIA) correlates with a lower incidence of myocardial infarction and ischemic stroke but an increased risk for hemorrhagic stroke. We describe mass spectrometric detection of the activation peptide variants in human serum. METHODS: We used differential peptide display (DPD) to compare comprehensive peptide maps from pairs of serum samples from healthy volunteers. Peptides were separated by liquid chromatography, and fractions were subjected to mass spectrometry. Mass spectra of all fractions were combined, giving a peptide map representing a two-dimensional display of peptide masses. After comparison of peptide mass maps, peptides that differentiated FXIIIA phenotypes were identified by mass spectrometry. RESULTS: Val34Leu polymorphisms of the activation peptide of FXIIIA were identified in 20 serum samples from 10 volunteers by DPD, and their sequences were confirmed by nanoelectrospray-ionization quadrupole time-of-flight mass spectrometry. Analysis of three (V34V, V34L, and L34L) phenotypes was confirmed by allele-specific genotypic analysis in all (n = 10) volunteers. CONCLUSION: DPD provides a simple and easy-to-use phenotype assay with advantages over PCR-based assays in being faster and directly analyzing the compound of interest.     

Rapid and accurate detection of the CFTR gene mutation 1811+1.6 kbA>G by real-time fluorescence resonance energy transfer PCR

The CFTR gene mutation 1811+1.6 kbA>G has been reported as associated with a severe phenotype of cystic fibrosis with pancreatic insufficiency. This mutation has been identified as a rather common one in the South West of France and in the Iberian Peninsula. Because of the precise geographical origin of the subjects and its frequency, the mutation has to be investigated with accuracy. We have developed an original real-time Fluorescence Resonance Energy Transfer (FRET) PCR assay for genotyping the mutation 1811+1.6 kbA>G. It is based on the amplification of a region spanning the mutation with simultaneous detection of the amplicon by hybridization with a bi-probe followed by a melting curve analysis. The results obtained are identical with those resulting from either restriction fragment length polymorphism analysis or sequencing. The distinction between the wild type and the mutation 1811+1.6 kbA>G is easy because the corresponding melting points shows a difference of 6 or 9.5 degrees C depending on the associated SNP A/T located 16 bp downstream. We demonstrated that a FRET assay showed enough sensitivity to discriminate between two nucleotide polymorphisms (SNPs) in the sequence of the sensor. In conclusion, this method is specific, fast, easy to perform, reproducible, inexpensive as it uses only one bi-probe and well adapted to daily practice.     

Rapid detection of point mutations by fluorescence resonance energy transfer and probe melting curves in Candida species

BACKGROUND: The LightCycler(TM) combines rapid amplification of nucleic acids in glass capillaries with melting curve analysis based on fluorescence resonance energy transfer for the sensitive detection of point mutations in various settings, such as drug resistance and hereditary diseases. Point mutations leading to an altered structure of lanosteroldemethylase, the target enzyme of the fungistatic azoles, are an important mechanism of acquired resistance in Candida albicans. METHODS: We screened 13 fluconazole-resistant C. albicans and 21 fluconazole-resistant C. tropicalis strains (minimum inhibitory concentration >128 mg/L), isolated from patients with AIDS, for the presence of defined point mutations by comparing conventional cycle sequencing with a newly designed LightCycler-based assay. RESULTS: In C. tropicalis, 5 of 21 isolates showed the wild-type sequence, and 8 of 21 showed the homozygous nucleotide exchange thymine to cytosine at position 1554 (T1554C). A heterozygous genotype was detected in 8 of 21 isolates by the LightCycler, but in only 3 of 21 isolates by conventional cycle sequencing. In 2 of 13 C. albicans isolates, a homozygous point mutation leading to an amino acid exchange at position 464 (glycine to serine) was detected in both assays. CONCLUSION: The LightCycler technique offers standardized, fast, sensitive, and reproducible detection of point mutations in different Candida spp.     

Rapid genotyping of HLA-B27 among Korean population by real-time PCR melting curve analysis

BackgroundReal-time PCR and melting curve analysis is the relatively recent method for HLA-B27 genotyping, which has advantages of being simple and rapid.MethodsThe accuracy of melting curve analysis for HLA-B27 was assessed in 153 clinical samples and 52 DNA samples from International Histocompatibility Workshop (IHW) cell lines, with sequence-based typing (SBT) as the reference method. We predicted melting reaction for various HLA-B27 subtypes using simulation software.ResultsFor clinical samples, 53 HLA-B27-positive and 100 negative results by melting curve analysis were confirmed by completely concordant SBT results. The B*27:05 allele was found in 50 patients, and the B*27:04 allele in 3 patients. Among 62 known alleles, 21 alleles had differences in the target sequence, including 10 alleles having mismatches in the primer binding site. In these alleles, differences in melting points (T_m) were predicted to be ≤ 1.2 °C. The predicted results were obtained when IHW samples were tested, which revealed slight lower T_m for B*27:06 and negative results for B*27:07.ConclusionsGenotyping of HLA-B27 by melting curve analysis was fast and reliable for routine laboratory testing for frequent alleles. In silico melting simulations provided useful information about the utility and limitation of this method for diverse HLA-B27 alleles.     

Rapid typing of the codon 129 polymorphism of the human prion protein gene by combined real-time PCR and melting curve analysis

Homozygosity of methionine (m/m) at amino acid residue 129 (codon 129) of the human prion protein (PrP) has been reported for all so far analyzed cases of the new variant Creutzfeldt-Jakob disease (vCJD). This contrasts with its general distribution in the healthy Caucasian population of only about 43%. For this reason a predisposition for carriers of the corresponding genotype to develop vCJD after infection with the bovine spongiform encephalopathy (BSE) agent is assumed, and PCR based methods such as allele-specific oligonucleotide hybridization or restriction analysis and sequencing have been developed for codon 129 genotyping. These methods are cumbersome and time-consuming and the need for extensive post-amplification manipulations increases the risk of carry-over contamination with amplified products. To overcome these shortcomings, the authors developed a real-time PCR assay on the LightCycler (LC) instrument combining PCR and temperature melting curve analysis (Tm) in a closed vessel format. Forty-six swabs and blood samples from healthy donors were tested. Of these 23 (50%) were heterozygous at codon 129, 4 (8.7%) homozygous for valine and 19 (41.3%) homozygous for methionine. Accuracy of LC-genotyping was confirmed by automated sequencing of the amplified products. Taken together, genotyping of the codon 129 polymorphism by combined LC-PCR and melting-curve analysis with the LC-instrument is a reliable and easy to perform method even in a screening context with numerous samples. Results can be obtained within 2 hours, including sample preparation.     

A simple and rapid genotyping assay for simultaneous detection of two ADRB2 allelic variants using fluorescence resonance energy transfer probes and melting curve analysis

Allelic variants at codons 16 and 27 of the beta(2)-adrenergic receptor gene (ADRB2) have shown clinical and pharmacological implications in asthma, hypertension, ischemic heart failure, diabetes, obesity, and cystic fibrosis. We have developed a simultaneous genotyping assay for the c.46A>G and c.79C>G allelic variants using hybridization probes and melting curve analysis. The assay was optimized on a panel of 30 DNA samples of known ADRB2 genotype as determined by sequencing with 100% concordance between the two techniques. Melting temperature (Tm) ranges for the different genotypes were obtained using data from three independent experiments. Single peaks for p.Arg16Arg (Tm = 57.76 degrees C +/- 0.10 degrees C) and p.Gly16Gly (Tm = 66.73 degrees C +/- 0.18 degrees C) and two melting peaks for p.Arg16Gly were obtained. Similarly, single peaks for p.Gln27Gln (Tm = 53.98 degrees C +/- 0.19 degrees C) and p.Glu27Glu (Tm = 64.93 degrees C +/- 0.16 degrees C) and two peaks for p.Gln27Glu were detected. Independent operators easily assigned genotypes in a sample set of 385 asthmatic patients. Haplotype and allele frequencies were in concordance with previously published data: Arg allele frequencies in children/adults were 0.34/0.30 in Caucasians and 0.45/0.52 in African Americans, and Gln allele frequencies were 0.58/0.52 in Caucasians and 0.82/0.84 in African Americans. Thus, the ADRB2 genotyping assay represents a highly reliable and rapid technique for routine clinical use in the simultaneous detection of ADRB2 variants.     

High-speed detection of the two common paraoxonase polymorphisms Leu55-->Met and Gln192-->Arg by real-time fluorescence PCR and melting curves.

Human paraoxonase (PON1) is a calcium-dependent esterase exclusively bound to apolipoprotein A-I and clusterin, containing high-density lipoprotein (HDL) particles that hydrolyzes organophosphates and aryl esters. Several studies have indicated that PON1 can prevent low-density lipoprotein (LDL) oxidation by hydrolyzing lipid peroxides in the lipoprotein, which is the crucial first step for atherogenesis. Therefore it may protect against the development of atherosclerosis. Serum PON1 activity has been shown to be decreased in familiar hypercholesterolemia and in diseases that are associated with accelerated atherogenesis. The PON1 gene has two common coding region polymorphisms, Leu55-->Met and Gln192-->Arg. Both polymorphisms have been identified as independent risk factors for cardiovascular disease in diabetic and non-diabetic patients. We have established high-speed and easy-to-perform genotyping for the two most significant PON1 gene polymorphisms, employing the LightCycler technology and melting curves. This technique eliminates PCR contamination related to sample handling and does not require digestion of PCR products with restriction enzymes and/or fragment separation on gels.     

Rapid detection of the irinotecan‐related UGT1A1*28 polymorphism by asymmetric PCR melting curve analysis using one fluorescent probe

BackgroundDetermination of UGT1A1 (TA)_n polymorphism prior to irinotecan therapy is necessary to avoid severe adverse drug effects. Thus, accurate and reliable genotyping methods for (TA)_n polymorphism are highly desired. Here, we present a new method for polymerase chain reaction (PCR) melting curve analysis using one fluorescent probe to discriminate the UGT1A1*1 [(TA)₆] and *28 [(TA)₇] genotypes.MethodsAfter protocol optimization, this technique was applied for genotyping of 64 patients (including 23 with UGT1A1*1/*1, 22 with *1/*28, and 19 with *28/*28) recruited between 2016 and 2021 in China‐Japan Friendship Hospital. The accuracy of the method was evaluated by comparing the results with those of direct sequencing and fragment analysis. The intra‐ and inter‐run precision of the melting temperatures (T_ms) were calculated to assess the reliability, and the limit of detection was examined to assess the sensitivity.ResultsAll genotypes were correctly identified with the new method, and its accuracy was higher than that of fragment analysis. The intra‐ and inter‐run coefficients of variation for the T_ms were both ≤0.27%, with standard deviations ≤0.14°C. The limit of detection was 0.2 ng of input genomic DNA.ConclusionThe developed PCR melting curve analysis using one fluorescent probe can provide accurate, reliable, rapid, simple, and low‐cost detection of UGT1A1 (TA)_n polymorphism, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.     

Asymmetric real-time PCR detection of BRCA1 5382insC mutation by melting curve analysis in the LightCycler

BACKGROUND: 5382insC BRCA1 frameshift mutation is a common founder mutation for many populations worldwide and a high-risk allele for the development of hereditary breast and/or ovarian cancer. Our goal was to develop a novel, reliable and rapid method for its detection. METHODS: We developed an asymmetric real-time PCR method with hybridization probes in the LightCycler. Genotyping was performed by melting curve analysis. RESULTS AND CONCLUSIONS: The developed method was in concordance with reference methods when tested in 85 peripheral blood and 107 tumor DNA samples from Greek breast and/or ovarian cancer patients. The described method proved to be simple, cost-effective, easy to perform and rapid enough for routine use as a screening method in high-risk families and especially in the Greek, Slavic and Jewish populations where 5382insC mutation is the most common BRCA1 mutation.     

Quantitative and qualitative analyses of the SNRPN gene using real-time PCR with melting curve analysis

Prader-Willi syndrome and Angelman syndrome are distinct neurodevelopmental disorders that are associated with the deletion of the chromosomal 15q11-13 region or uniparental disomy of chromosome 15. In this article, we applied SYBR Green I-based real-time PCR and melting curve analysis assay for rapid genotyping of the small nuclear ribonucleoprotein polypeptide N (SNRPN) gene methylation status and for detecting aberrations in copy number in a single tube. A single pair of primers was designed to create a 357 bp fragment containing the cytosine phosphodiester guanine islands in the SNRPN promoter and to amplify both unmethylated and methylated sequences. Genotypes were identified based on the TC value for copy number changes and the characteristic melting temperature of methylated cytosine phosphodiester guanine. Genotyping of SNRPN was performed on blood samples of 20 individuals with Prader-Willi syndrome, 3 individuals with Angelman syndrome, and 20 unaffected individuals. The promoter methylation status and the copy number changes were successfully determined and compared with standard methylation-specific PCR, and were validated by multiplex ligation-dependent probe amplification. This single-tube, SYBR Green I, real-time PCR with melting curve assay is rapid, reliable, sensitive, and easy to perform. It is suitable for high-throughput analysis as an alternative technique for quantitative and qualitative analysis of target genes.     

Rapid detection of K-ras mutations in bile by peptide nucleic acid-mediated PCR clamping and melting curve analysis: comparison with restriction fragment length polymorphism analysis

BACKGROUND: Current methods for detection of K-ras gene mutations are time-consuming. We aimed to develop a one-step PCR technique using fluorescent hybridization probes and competing peptide nucleic acid oligomers to detect K-ras mutations in bile and to compare the efficacy with restriction fragment length polymorphism (RFLP) analysis. METHODS: Bile samples were obtained from 116 patients with biliary obstruction, including gallstones (n = 64), benign biliary strictures (n = 6), pancreatic cancer (n = 20), and cholangiocarcinoma (n = 26). The DNA was extracted and subjected to K-ras mutation analysis by real-time PCR and RFLP analysis. Mutations were confirmed by direct sequencing. The sensitivity and specificity were calculated according to the clinical results. RESULTS: The analysis time for real-time PCR was <1 h, whereas RFLP analysis took more than 2 days. With the sensor probe designed for the GAT (G12D) mutant in codon 12 of the K-ras gene, the real-time PCR method also detected the GTT (G12V) mutant. In contrast, a specific sensor probe for the TGT (G12C) mutant detected GAT (G12D), AGT (G12S), and GTT (G12V) mutants in addition to the TGT mutant. The real-time PCR assay allowed the detection of mutation in a 3000-fold excess of wild-type bile DNA. In bile, K-ras codon 12 mutations were detected in 16 of 46 malignant cases by real-time PCR with the TGT probe and 15 by RFLP analysis. All benign cases were wild type. CONCLUSION: Real-time PCR with a cysteine-specific (TGT) sensor probe can rapidly detect K-ras gene mutations in bile and diagnose malignant biliary obstruction with high specificity.     

Rapid and accurate detection of Pseudomonas aeruginosa by real-time polymerase chain reaction with melting curve analysis targeting gyrB gene

Laboratory detection of Pseudomonas spp., particularly Pseudomonas aeruginosa, is an important assay in the nosocomial control. The study was designed firstly to establish a new assay-applied LightCycler polymerase chain reaction (PCR) technology with melting curve analysis (MCA). A total of 224 Gram-negative isolates were used to verify the assay system. The PCR with MCA method using the P. aeruginosa-specific gyrase B gene primers was rapid and accurate; the total run is approximately 3 h, and the sensitivity and specificity relative to the Vitek (bioMerieux, Hazelwood, MO) results were 98.1% and 100%, respectively. Vitek identification system was not able to identify the isolates from the new Pseudomonas otitidis spp. opposite to the real-time PCR. This assay was validated to be accurate with an overall sensitivity and specificity of 98.7% and 98.9%, respectively. Conclusively, this rapid and accurate PCR assay with MCA will help to manage and control infections with P. aeruginosa.     

Rapid detection of the SPINK5 polymorphism Glu420Lys by real-time PCR technology

BACKGROUND: The SPINK5 gene, encoding the serine protease inhibitor LEKTI maps to chromosome 5q32, and has been suggested to be a locus predisposing to atopic diseases in general. The Glu420Lys variant showed significant association with atopy, asthma and atopic dermatitis in recent studies. AIMS OF THE STUDY: Development of a high throughput assay to analyse the polymorphism G1258A (Glu420Lys) in exon 14 of the SPINK5 gene followed by the validation using samples of 235 latex-allergic health care workers (HCWs) with (N=63) and without asthma (N=172), and 80 non-atopic controls. METHODS: Twenty DNA samples were first analysed by a polymerase chain restriction fragment analysis (RFLP) using Hph I to generate defined control DNAs which were used for the development of the assay suitable for the detection of the Glu420Lys variant by LightCycler technology. RESULTS AND CONCLUSIONS: 315 samples were successfully screened with this new assay. The temperatures in the melting analysis of the SPINK5 exon 14 PCR product were characteristic to the probes hybridised to the mutant (AA) at 51.5 degrees C and to the wild-type (GG) at 59.5 degrees C. The fast and reliable mutation detection in the tested samples makes this high-speed method suitable for larger epidemiological studies.     

Factor XIII Val34Leu polymorphism and γ-chain cross-linking at the site of microvascular injury in healthy and coumadin-treated subjects

Fibrin (Fn) cross-linking by activated factor (F) XIII is essential for clot stability. In vitro, a common Leu34 polymorphism of the FXIIIA-subunit increases the rate of thrombin-mediated FXIII activation, but not cross-linking activity upon complete FXIII activation. The effect of FXIII Val34Leu polymorphism on fibrin(ogen) cross-linking in vivo when vascular injury triggers the blood coagulation has not been studied yet. Using quantitative immunoblotting with antibodies raised against FXIIIA-subunits, fibrinogen, and gamma-gamma-dimers, the rates of FXIIIA cleavage and fibrin(ogen) cross-link formation in the fluid phase of 30-s blood samples collected at the site of microvascular injury were compared in the Leu34-positive and -negative healthy individuals and patients on long-term oral anticoagulation. In addition to accelerated FXIII activation, in healthy subjects the presence of FXIII Leu34 allele was associated with increased soluble gamma-gamma-dimer formation by 40% (1355 +/- 17 microg L(-1) for Leu34 carriers vs. 804.3 +/- 17 microg L(-1) for Leu34 non-carriers; P = 0.028) at the site of microvascular injury. This solution phase effect was abolished in coumadin-treated patients (369.4 +/- 75.9 microg L(-1) for Leu34 carriers vs. 290.5 +/- 35.9 microg L(-1) for Leu34 non-carriers; P > 0.05). The present study indicates that the Leu34 allele affects soluble gamma-gamma-dimer formation in untreated individuals, but not in those receiving acenocoumarol. Our data may help elucidate the impact of the FXIII Val34Leu polymorphism on Fn cross-linking in vivo and its modulation by oral anticoagulants.     

Vascular endothelial growth factor (VEGF) polymorphisms in HELLP syndrome patients determined by quantitative real-time PCR and melting curve analyses

BACKGROUND: The vascular endothelial growth factor (VEGF) has a critical role in vasculogenesis and vascular permeability in several diseases including preeclampsia. There are at least 30 single nucleotide polymorphic (SNP) places on this gene. VEGF G+405C, C-2578A and C-460T SNPs are known to be related to VEGF production. VEGF polymorphisms were studied in preeclampsia, but not in HELLP syndrome. Therefore, we decided to determine the allele and genotype frequencies of VEGF G+405C, C-460T and C-2578A SNPs in healthy pregnant women and HELLP syndrome patients. METHODS: The authors introduced a quantitative real-time PCR method for the determination of the three VEGF SNPs. Blood samples were collected from 71 HELLP syndrome patients and 93 healthy controls. DNA was isolated by using silica adsorption method. The SNPs were determined by quantitative real-time PCR and melting curve analysis using LightCycler. RESULTS: There were significant differences in the allele and genotype frequencies of VEGF C-460T SNP between the two study groups. The T allele was present in 71.1% in the HELLP group, while in 53.8% in the controls (p=0.0014). The TT genotype occurred significantly more frequently in the HELLP group than in the control group (45.1% vs. 21.5%; p (for genotype frequencies)=0.0011). The TT genotype carriers had an increased risk of HELLP syndrome, which was independent of maternal age and primiparity (adjusted odds ratio (OR)=3.03, 95% confidence interval (CI)=1.51-6.08; p=0.002). Although the VEGF G+405C allele and genotype distributions did not differ significantly between the two groups, the CC genotype carriers were also found to have an increased risk for HELLP syndrome after adjustment for maternal age and primiparity (adjusted OR=3.67, 95% CI=1.05-12.75; p=0.041). The VEGF C-2578A SNP was not associated with HELLP syndrome. CONCLUSIONS: The quantitative real-time PCR combined with melting curve analyses is a fast and reliable method for the determination of VEGF SNPs. We found that the VEGF -460TT and +405CC genotype carriers have an increased risk of HELLP syndrome. As these two SNPs were previously observed to be related to production of the VEGF protein, we suppose that these VEGF polymorphisms -- interacting with other genetic and environmental factors - could play a role in the development of HELLP syndrome.